Triple transgenic mouse model of alzheimer&#39;s disease

ABSTRACT

The present invention relates to a triple transgenic animal model for Alzheimer&#39;s disease as well as to methods for generating multi-transgenic animals. The present invention also relates to methods for screening biologically active agents potentially useful for treating and/or ameliorating Alzheimer&#39;s disease (AD) or AD-type pathologies, compositions useful for treating AD or AD-type pathologies, and methods of treating AD patients.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application Ser.No. 60/343,383, entitled “Triple Transgenic Mouse Model of Alzheimer'sDisease,” filed Dec. 20, 2001, the contents of which are herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The neuropathology of Alzheimer's Disease (AD) is characterized by twohallmark lesions: diffuse and neuritic plaques, which are predominantlycomposed of the amyloid-β (Aβ) peptide, and neurofibrillary tangles,which are composed of filamentous aggregates of hyperphosphorylated tauprotein (Selkoe, D. J. (2001) Alzheimer's disease: genes, proteins, andtherapy, Phlysiol. Rev. 81, 741-66). Loss of neuronal synaptic densityand synapse number represent another invariant feature of the diseasethat appears to precede overt neuronal degeneration (DeKosky, S. T. &Scheff, S. W. (1990) Synapse loss in frontal cortex biopsies inAlzheimer's disease: correlation with cognitive severity. Ann. Neurol.27,457-64; Scheff, S. W., Scott, S. A. & DeKosky, S. T. (1991)Quantitation of synaptic density in the septal nuclei of young and agedFischer 344 rats. Neurobiol. Aging 12, 3-12).

Notably, the memory and cognitive decline observed in AD patientscorrelates better with the synaptic pathology than either plaques ortangles (Terry, R. D. et al. (1991) Physical basis of cognitivealterations in Alzheimer's disease: synapse loss is the major correlateof cognitive impairment, Ann. Neurol. 30, 572-80; Dickson, D. W. et al.(1995) Correlations of synaptic and pathological markers with cognitionof the elderly, Neurobiol. Aging 16, 285-98; Sze, C. I. et al. (1997)Loss of the presynaptic vesicle protein synaptophysin in hippocampuscorrelates with cognitive decline in Alzheimer disease, J. Neuropathol.Exp. Neurol. 56, 933-44; Masliah, E. et al. (2001) Altered expression ofsynaptic proteins occurs early during progression of Alzheimer'sdisease, Neurology 56, 127-9), and is likely the most significant factorcontributing to the initial stages of memory loss (Selkoe, D. J. (2002)Alzheimer's disease is a synaptic failure, Science 298, 789-91).

Gene-targeted and transgenic mice have proven to be invaluable foraddressing some of the mechanisms underlying the synaptic dysfunction(Larson, J., et al. (1999) Alterations in synaptic transmission andlong-term potentiation in hippocampal slices from young and aged PDAPPmice, Brain Res. 840, 23-35; Hsia, A. Y. et al. (1999)Plaque-independent disruption of neural circuits in Alzheimer's diseasemouse models, Proc. Natl. Acad. Sci. U.S.A. 96, 3228-33; Chapman, P. F.et al. (1999) Impaired synaptic plasticity and learning in aged amyloidprecursor protein transgenic mice, Nat. Neurosci. 2, 271-6; Fitzjohn, S.M. et al. (2001) Age-related impairment of synaptic transmission butnormal long-term potentiation in transgenic mice that overexpress thehuman APP695SWE mutant form of amyloid precursor protein, J. Neurosci.21, 4691-8), although none of these models recapitulate both hallmarkpathological lesions (Wong, P. C., et al. (2002) Genetically engineeredmouse models of neurodegenerative diseases, Nat. Neurosci. 5, 633-9).What is needed, therefore, is an animal model transgenic for multipleAlzheimer-related genes that produces multiple major features associatedwith this disease, including both hallmark pathological lesions.

In generating a transgenic animal such as a transgenic mouse, a humantransgene is typically microinjected into fertilized eggs from a normal,nontransgenic mouse. Such transgenic mice have, in turn, been used togenerate “double” transgenic mice by mating different strains oftransgenic mice, each containing a different transgene, to produce aline containing both transgenes (i.e., a double transgenic mouseline)(See, e.g., U.S. Pat. No. 5,898,094). Similarly, a doubletransgenic mouse can be bred with a transgenic mouse containing a thirdtransgene to generate a triple transgenic mouse.

The disadvantages of this process of breeding triple transgenic mice areseveral. First, it is exceedingly time consuming to produce a multipletransgenic mouse by a series ofmicroinjection/breeding/screening/selection steps as described above.Second, this process is very costly as it requires extensive breeding,housing, screening, and personnel costs. Third, this process producesmice with a variable genetic background, which can be a major problemfor therapeutic and behavioral investigations.

What is also needed, therefore, is a process for producing multipletransgenic mice that avoids one or more of the above-listeddisadvantages.

SUMMARY OF THE INVENTION

One aspect of the invention is directed to a triple transgenic animalmodel for Alzheimer's disease.

Another aspect of the invention is directed to methods for generatingmulti-transgenic animals.

Another aspect of the invention is directed to methods for screeningbiologically active agents potentially useful for treating and/orameliorating Alzheimer's disease (AD) or AD-type pathologies.

Another aspect of the invention is directed to compositions useful fortreating AD or AD-type pathologies.

Another aspect of the invention is directed to methods of treating ADpatients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating the design of a novel strategy for thegeneration of the 3×Tg-AD mouse model. Single cell embryos wereharvested from mutant homozygous PS1_(M146V) knockin mice. Using thepronuclear microinjection technique, two independent transgenicconstructs encoding human APP_(Swe) and tau_(P301L) (4R/0N), under thecontrol of the mouse Thy1.2 regulatory elements, were co-injected. Theentire mouse Thy1.2 genomic sequence is shown with exons depicted asboxes and noncoding sequences as thin lines. The injected embryos werere-implanted into foster mothers and the resulting offspring genotypedto identify 3×Tg-AD mice.

FIG. 2 is a chart with data showing the success of the novel strategyfor producing triple transgenic mice.

FIG. 3 is an autoradiogram of a Western Blot demonstrating expression ofhuman tau protein in triple transgenic mice. TAUC17 is an antibody thatdetects both human and mouse Tau protein. HT7 is an antibody that isspecific for human Tau protein.

FIG. 4 is an autoradiogram of a Western Blot demonstrating expression ofhuman APP protein in triple transgenic mice. 22C11 is an antibody thatdetects both human and mouse APP protein. 6E10 is an antibody that isspecific for human APP protein.

FIG. 5 is an autoradiogram of a Western blot demonstrating expression ofC99 protein in brain tissue of triple transgenic mice.

FIG. 6A is an autoradiogram of a Western blot demonstrating expressionof Aβ protein in a triple transgenic mouse. FIG. 6B is a chart showingELISA results further demonstrating Aβ expression in triple transgenicmice.

FIG. 7A is a Southern Blot showing DNA levels in selected candidatehomozygous mice. FIG. 7B is a chart containing data showing successfulbreeding to homozygosity.

FIG. 8A is a Western blot showing that homozygous triple transgenic miceexpress both human TAU and human APP at twice the levels of hemizygousmice. β actin is shown as a control.

FIG. 8B is a representative Southern blot comparing the gene dosage ofthe tau and APP transgenes from tail DNA of hemizygous and homozygousmice. FIG. 8C is an immunoblot comparing steady state levels of humanAPP and tau proteins in the brains of 4-month old hemizygous andhomozygous 3×Tg-AD mice. For both APP (detected with antibody 22C11) andtau (detected with antibody tau5), the levels are doubled in thehomozygous mice. FIG. 8D is a bar graph showing steady state levels ofthe βAPP and tau protein are approximately 3-to-4-fold and 6-to-8 foldhigher than endogenous levels in hemizygous and homozygous mice,respectively.

FIG. 9 is a photograph of sections of brains from a triple transgenicmouse (B1) and a control mouse (PS1-KI), stained with hematoxilin andeosin. CA3 is a region of the hippocampus, while DG is the DentateGyrus, another region of the brain. The arrows point to a pathologicalalteration, a tangle-like pathology, found in the brain sections takenfrom triple transgenic mice.

FIG. 10 is a photograph of brain sections taken from a triple transgenicmouse and stained with 6E10, an antibody specific for human APP protein.The black areas indicate another pathological alteration in the brainsof triple transgenic mice; namely, Aβ deposition.

FIG. 11 shows an analysis of steady-state levels of the human transgenesin 3×Tg-AD mice. (A) Multiple peripheral tissues were surveyed byimmunoblot analysis to determine the transgene expression profile. Tauand APP appear to be exclusively expressed in the central nervous system(CNS). (B) Quantitative comparison of transgene products in variousbrain subregions by western blotting. The hippocampus and cerebralcortex, two prominent AD-afflicted regions, are among the brain regionscontaining the highest steady-state levels of the human transgeneproducts. (C) Aβ levels are doubled in the homozygous mice.Immunoprecipitation/western blotting shows that 4-KDa Aβ is detectablein the brains of both hemizygous and homozygous mice. (D) Aβ40 and Aβ42levels measured by ELISA from different aged mice (n=3/group). Levelswere measured as described previously (Parent, A., et al. (1999)Synaptic transmission and hippocampal long-term potentiation intransgenic mice expressing FAD-linked presenilin 1, Neurobiol. Dis. 6,56-62). Non-transgenic (NonTg), hemizgyous and homozygous mice aredepicted as blue, yellow, and red bars, respectively. Statisticaldifference between hemizygous and NonTg mice is denoted by **, whereas *indicates that homozygous mice are statistically different fromhemizygous and NonTg mice.

FIG. 12. Aβ deposition precedes tau pathology in 3×Tg-AD mice. (A)Aβ-immunoreactivity is first detected intracellularly in neurons withinthe neocortex. (B, C) Low and high magnification views, respectively, ofthe neocortex from a 9 month old homozygous mouse showing extracellularAβ deposits in layer 4-5 of the neocortex. (D-F) Hippocampi ofhomozygous 3×Tg-AD mice (6, 12, 15 months old, respectively) showingfirst intraneuronal Aβ staining in the pyramidal neurons in the CA1region (D) and then with extracellular Aβ staining (E,F). (G-I) Humantau immunoreactivity, detected with the human specific anti-tauantibody, HT7, is first apparent in the hippocampus and becomes moresevere with advancing age (6, 12, 15 months old, respectively). PanelsA-F show Aβ immunoreactivity using monoclonal antibody 6E10, panels G-Ishow tau immunostaining with antibody HT7. Original magnifications, 5×(D-I), 10× (B), 20× (A,C).

FIG. 13. Tau pathology initiates in the hippocampus and progresses tothe neocortex. (A,B) Low and high magnification views of the hippocampusshowing human tau immunopositive pyramidal neurons following stainingwith the conformational specific antibody MC1. (C) High magnificationview showing immunopositive neurons following staining with antibodyAT8, which detects phosphorylated tau proteins at serine 202 andthreonine 205 residues. (D,E) Low and high magnification views of thehippocampus showing tau immunoreactive pyramidal neurons followingstaining with antibody AT180, which detects phosphorylated tau proteinsat threonine 231 residues. (F) Immunostaining of neocortex with thehuman specific tau antibody HT7. (G,H) High magnification view ofneocortical brain region from 12 month-old homozygous mice stained withGallyas silver stain and thioflavin S. (I) High magnification view ofthe neocortex showing Aβ and tau co-localized to many of the samepyramidal neurons. Aβ was used immunostained with antibody 6E10 followedby detection with true blue (blue staining), whereas tau wasimmunostained with antibody HT7 and detected using DAB (brown staining).(J) GFAP immunoreactive astrocytes are also present around extracellularAB deposits. Original magnifications, 5× (A,D), 10× (P), 20× (C,I,J),40× (B,E), 100× (G,H).

FIG. 14. Age related synaptic dysfunction in 3×Tg-AD mice. (A)Input/output (I/O) curves and representative fEPSPs at increasingstimulus strengths are shown for NonTg, PS1_(M46V) KI, and 3×Tg-AD mice,showing no differences at one month of age. NonTg, 1.42±0.17 mV/ms, n=12slices from 6 mice; PS1 KI, 0.60±0.06 mV/ms, in n=14/8, P<0.001;3×Tg-AD, 0.53±0.06 mV/ms, n=14/6, P<0.001. (B) Smaller fEPSPs are evokedin six month PS1_(M146V) KI and 3×Tg-AD mice as compared with NonTgmice, indicating impaired synaptic transmission. (C) Paired-pulsefacilitation (PPF) was measured at an interpulse interval of 50 ms andwas normal for all groups at one month of age. (D) At six months of age3×Tg-AD mice exhibited normal PPF compared to NonTg mice but PS1_(M146V)KI mice exhibited significantly enhanced PPF. (E)fEPSP slopes wererecorded and were expressed as the percentage of the pre-tetanusbaseline. Representative fEPSPs before (solid line) and 60 minutes afterthe induction of LTP (dotted line) are shown. LTP was normal in allgroups at one month of age. (F) LTP was markedly impaired in 3×Tg-ADmice. In contrast, short-term LTP was enhanced in PS1_(M146V) KI mice,but was otherwise normal. The amount of potentiation of fFPSPs between0-10 minutes after HFS was 232±13% in NonTg mice (n=12 slices, 6animals) and was significantly higher in PS1_(M146V) KI mice (282±19%,n=14 slices, 8 animals, P<0.05) but was significantly reduced in 3×Tg-ADmice (143±6%, n=14 slices, 6 animals, P<0.001). The amount ofpotentiation between 50-60 minutes after MFS was 190±11% in NonTg miceand was not significantly different in PS1_(M) _(146V) KI Mice (212±13%,P<0.1), but was significantly reduced in 3×Tg-AD mice (125±8%, P<0.001).

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the celllines, constructs, and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

Definitions

The term “transgene” refers to the genetic material which has been or isabout to be artificially inserted into the genome of an animal,particularly a mammal and more particularly a mammalian cell of a livinganimal.

“Transgenic animal” refers to a non-human animal, usually a mammal,having a non-endogenous (i.e., heterologous) nucleic acid sequencepresent as an extrachromosomal element in a portion of its cells orstably integrated into its germ line DNA (i.e., in the genomic sequenceof most or all of its cells). Heterologous nucleic acid is introducedinto the germ line of such transgenic animals by genetic manipulationof, for example, embryos or embryonic stem cells of the host animal.

“Operably linked” means that a DNA sequence and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate transcriptional activator proteins are bound to theregulatory sequence(s). For example, a nucleic acid sequence encodinghuman amyloid-β precursor protein (βAPP) may be operably linked to amouse Thy1.2 promoter to facilitate production of βAPP polypeptide inmouse cells.

“Aβ pathology” refers to diffuse and neuritic plaques observed in thebrain, which are predominantly composed of the amyloid-β(Aβ) peptide. Aβpathology also includes neuronal and/or glial inclusions or insolubledeposits that stain positively with anti-Aβ antibodies.

“Tau pathology” refers to neurofibrillary tangles observed in the brain,including one or more of paired helical filaments (PHFs), straight Taufilaments, and any other type of Tau filament. Tau pathology alsoincludes neuronal and/or glial inclusions or insoluble deposits thatstain positively with anti-Tau antibodies.

“AD pathology” refers to pathological changes in AD patients, forexample, Aβ pathology, Tau pathology, loss of neuronal synaptic densityand synapse number, decreased cognitive ability, memory loss and otherpathologies associated with AD.

“AD-associated polypeptides” refers to polypeptides, wild-type ormutant, known to be associated with AD and AD-related pathologies, suchas Aβ pathology and Tau pathology. AD-associated polypeptides include,but are not limited to, Presenilin, βAPP, and Tau.

“Heterologous polypeptides” refers to the polypeptides, wild-type ormutant, expressed by transgenes in a transgenic animal. Examples ofheterologous polypeptides may include human Presenilin, Tau, βAPP andother AD-associated polypeptides expressed in a non-human animal.

Introduction

One aspect of the instant invention is directed to a triple transgenicanimal model of Alzheimer's disease. In one example, two human geneswere inserted into the genome of a genetically-altered mouse alreadycontaining a human gene, thereby producing a triple transgenic mouse.

Another aspect of the instant invention is directed to a process forproducing a multi-transgenic animal in general. Typically, transgenicmice are generated by microinjecting a foreign gene into fertilized eggsisolated from a normal, nontransgenic, mouse. In the instant invention,it has been shown that it is possible to create a mouse expressingadditional human transgenes by starting with a mouse that is alreadytransgenic. That is, single-cell embryos (fertilized eggs) from anexisting transgenic mouse have been harvested and additional transgeneDNA fragments have been microinjected into the cells. The results shownherein demonstrate that existing fertilized eggs from transgenic micecan withstand the microinjection process and that it is possible tosimultaneously co-microinject more than one type of transgene DNAfragment to successfully produce a multi-transgenic mouse.

The advantage to this process is that it permits the production of amulti-transgenic mouse on the same genetic background. In addition, itis possible to readily and easily breed multi-transgenic mice.Previously, the generation of a multi-transgenic mouse requiredextensive breeding strategies and led to the production of mice with amixed genetic background, a confounding variable in research aimed atbehavioral, pharmacological and therapeutic studies.

Although the process for generating a multi-transgenic animal isdescribed herein with reference to a multi-transgenic mouse model forAlzheimer's disease, one of skill in the art will appreciate that thedescribed process may be used to generate multi-transgenic animals otherthan mice, for example, rats, hamsters, rabbits, etc., using transgenesother than those related to AD and AD-type pathologies. The presentinvention of a method for generating multi-transgenic animals is notintended to be limited to the particular triple transgenic mouse modelof AD described below but rather may be applied to any non-human animalmodel in which it is desirable to express multiple transgenes.

The novel triple transgenic mouse model produced in the instantinvention contains the three major genes that contribute to the hallmarkpathological features of Alzheimer's disease. Starting with a transgenicmouse obtained from Dr. Mark Mattson of the NIH, this transgenic mousemodel was improved by the introduction of two additional transgenes thatencode human genes that contribute to Alzheimer's disease pathology,using a novel strategy that did not require crossing mouse lines. Thesemice are exceedingly valuable for therapeutic investigations and forbasic research aimed at understanding the behavioral, physiological,molecular/cell biological and pharmacological processes leading todementia in an animal model.

This novel triple transgenic model (3×Tg-AD) that is the firsttransgenic model to develop both Aβ and Tau pathology in AD-relevantbrain regions. 3×Tg-AD mice develop extracellular Aβ deposits prior totangle formation, consistent with the amyloid cascade hypothesis. Thesemice exhibit deficits in synaptic plasticity, including long-termpotentiation (LTP) that occurs prior to extracellular Aβ deposition andtau pathology, but associated with intracellular Aβ immunoreactivity.Such findings suggest that synaptic dysfunction is a proximal defect inthe pathobiology of Alzheimer's disease that precedes the development ofextracellular plaque formation and tau pathology. These 3×Tg-AD mice maynow be used to assess the efficacy of anti-Aβ therapies in mitigatingsynaptic dysfunction and tau-mediated neurodegeneration.

Transgenic Animals

Transgenic animals comprise exogenous DNA incorporated into the anima'scells to effect a permanent or transient genetic change, preferably apermanent genetic change. Permanent genetic change is generally achievedby introduction of the DNA into the genome of the cell. Vectors forstable integration include plasmids, retroviruses and other animalviruses, YACs, and the like. Generally, transgenic animals are mammals,most typically mice.

The exogenous nucleic acid sequence may be present as anextrachromosomal element or stably integrated in all or a portion of theanimal's cells, especially in germ cells. Unless otherwise indicated, atransgenic animal comprises stable changes to the germline sequence.During the initial construction of the animal, chimeric animals(chimeras) are generated, in which only a subset of cells have thealtered genome. Chimeras may then be bred to generate offspringheterozygous for the transgene. Male and female heterozygotes are maythen be bred to generate homozygous transgenic animals.

Typically, transgenic animals are generated using transgenes from adifferent species or transgenes with an altered nucleic acid sequence.For example, a human gene, such as the nucleic acid encoding βAPP orTau, may be introduced as a transgene into the genome of a mouse orother animal. The introduced gene may be a wild-type gene, naturallyoccurring polymorphism, or a genetically manipulated sequence, forexample having deletions, substitutions or insertions in the coding ornon-coding regions. For example, an introduced human βAPP gene may bewild type or may include a mutation such as the previously-characterized“Swedish mutation.” Where the introduced gene is a coding sequence, itis usually operably linked to a promoter, which may be constitutive orinducible, and other regulatory sequences required for expression in thehost animal.

Nucleic Acid Compositions

Constructs for use in the present invention include any constructsuitable for use in the generation of transgenic animals having thedesired levels of expression of a desired transgene as, for example,Presenilin-, Tau-, or βAPP-encoding sequence, other AD-associatedpolypeptide-encoding sequence, or other heterologouspolypeptide-encoding sequence. Methods for isolating and cloning adesired sequence, as well as suitable constructs for expression of aselected sequence in a host animal, are well known in the art. Inaddition to the heterologous polypeptide-encoding sequences, theconstruct may contain other sequences, such as a detectable marker.

The heterologous polypeptide-encoding construct can contain a wild-typesequence or mutant forms, including nucleotide insertions, deletions,splice variants, and base substitutions. An AD-associatedpolypeptide-encoding construct having nucleotide insertions, deletions,splice variants, or base substitutions associated with AD and/or AD-typepathologies in humans is one example of a useful construct.

The heterologous polypeptide-encoding construct may include the openreading frame encoding specific polypeptides, introns, and adjacent 5′and 3′ non-coding nucleotide sequences involved in the regulation ofexpression. The heterologous polypeptide-encoding portion of theconstruct may be cDNA or genomic DNA or a fragment thereof The genes maybe introduced into an appropriate vector for extrachromosomalmaintenance or for integration into the host.

The nucleic acid compositions used in the subject invention may encodeall or a part of the heterologous polypeptide-encoding sequence asappropriate. Fragments may be obtained of the DNA sequence by chemicallysynthesizing oligonucleotides in accordance with conventional methods,by restriction enzyme digestion, by PCR amplification, and by othertechniques known in the art.

The Presenilin-1 Mutant Knock-in Mouse

Mutations in the presenilin-1 (PS1) gene on chromosome 14 have beencausally linked to many cases of early-onset inherited AD. PS1 mutant“knock-in” mice (PS1_(M146V)), in which a homologous exon in the mousePS1 gene has been replaced with an AD-linked PS1 mutation, have beenpreviously described (Guo, Q. et al. (1999) Increased vulnerability ofhippocampal neurons to excitotoxic necrosis in presenilin-1 mutantknock-in mice, Nat. Med. 5, 101-6). In the homozygous state, such miceproduce only mutant PS1 and no wild-type PS1. Moreover, the mutant PS1has been “humanized” in these mice by the introduction of a secondmutation, an I145V substitution.

The APP Gene and its Derivatives Suitable for Use in the PresentInvention

The APP gene has been described in U.S. Pat. No. 6,455,757, the entirecontents of which are hereby incorporated by reference.

Transgenic animals of the present invention comprise a heterologoussequence encoding a desired APP gene, preferably a human βAPP gene.Preferably, the host animal produces high levels of human βAPP or itsproteolytic fragments, such as human Aβ₄₂. Preferably, the βAPP geneencodes a genomic βAPP sequence or a sequence encoding a spliced βAPPgene (e.g., a cDNA), more preferably a full-length human βAPP cDNAsequence. Alternatively, the βAPP gene can be a mutant, particularly anβAPP mutant associated with AD and/or an AD-type pathology. Mutants ofparticular interest include human βAPP cDNA harboring the Swedish doublemutation.

Several isoforms and homologs of βAPP are known. Additional homologs ofcloned βAPP are identified by various methods known in the art. Forexample, nucleic acids having sequence similarity are detected byhybridization under low stringency conditions. Labeled nucleotidefragments can be used to identify homologous βAPP sequences as, forexample, from other species.

The host animals can be homozygous, hemizygous or heterozygous for theβAPP-encoding sequence, preferably homozygous. The βAPP gene can also beoperably linked to a promoter to provide for a desired level ofexpression in the host animal and/or for tissue-specific expression.Expression of βAPP can be either constitutive or inducible.

βAPP genes suitable for use in the present invention have been isolatedand sequenced. The sequence for human β-amyloid precursor protein isfound at GenBank Accession No. XM047793. See also, Table 2 of U.S. Pat.No. 6,455,757, providing a list of human APP sequences with Genbankaccession numbers relating to the listed APP sequences.

The Tau Gene and its Derivatives Suitable for use in the PresentInvention

The tau gene encodes the microtubule associated protein Tau that is themajor component of the PHFs that make up the characteristic tangles seenin AD and other neurodegenerative disorders. The human Tau protein foundin brain is encoded by eleven exons. The sequence of the wild-type humantau gene is described by Andreadis, A. et al. (1992) Biochemistry,31:10626-10633. The sequence for the human tau gene is found at GenBankAccession No. 11426018.

Transgenic animals of the present invention comprise a heterologoussequence encoding a desired tau gene, preferably a human tau gene.Preferably, the host animal produces high levels of human tau or itsproteolytic fragments. Preferably, the tau gene encodes a genomic tausequence or a sequence encoding a spliced tau gene (e.g., a cDNA), morepreferably a full-length human tau cDNA sequence. Alternatively, the taugene can be a mutant, particularly a tau mutant associated with ADand/or an AD-type pathology. Mutants of particular interest includehuman tau cDNA harboring the P301L mutation. Other mutants of the taugene suitable for use in the present invention are described in U.S.Pat. No. 6,475,723, the entire contents of which are hereby incorporatedby reference.

Several isoforms and homologs of tau are known. Additional homologs ofcloned tau are identified by various methods known in the art. Forexample, nucleic acids having sequence similarity are detected byhybridization under low stringency conditions. Labeled nucleotidefragments can be used to identify homologous tau sequences as, forexample, from other species.

The host animals can be homozygous, hemizygous or heterozygous for theTau-encoding sequence, preferably homozygous. The tau gene can also beoperably linked to a promoter to provide for a desired level ofexpression in the host animal and/or for tissue-specific expression.Expression of tau can be either constitutive or inducible.

Methods of Making Transgenic Animals

Transgenic animals can be produced by any suitable method known in theart, such as manipulation of embryos, embryonic stem cells, etc.Transgenic animals may be made through homologous recombination, wherethe endogenous locus is altered. Alternatively, a nucleic acid constructis randomly integrated into the genome. Vectors for stable integrationinclude plasmids, retroviruses and other animal viruses, YACs, and thelike.

Specific methods of preparing the transgenic animals of the invention asdescribed herein. However, numerous methods for preparing transgenicanimals are now known and others will likely be developed. See, e.g.,U.S. Pat. Nos. 6,252,131, 6,455,757, 6,028,245, and 5,766,879, allincorporated herein by reference. Any method that produces a transgenicanimal expressing multiple AD-associated polypeptides is suitable foruse in the practice of the present invention. The microinjectiontechnique described is particularly useful for incorporating transgenesinto the genome without the accompanying removal of other genes.

Drug Screening Assays

The transgenic animals described herein may be used to identifycompounds useful in the treatment of AD and/or AD-related pathologies.For example, transgenic animals of the present invention may be treatedwith various candidate compounds and the resulting effect, if any, onthe formation of diffuse and neuritic plaques and neurofibrillarytangles, and/or the on the loss of neuronal synaptic density and synapsenumber evaluated. The effect of candidate compounds on cognition andmemory may also be evaluated in transgenic animals of the presentinvention, using techniques known in the art. Preferably, the compoundsscreened are suitable for use in humans.

Drug screening assays in general suitable for use with transgenicanimals are known. See, for example, U.S. Pat. Nos. 6,028,245 and6,455,757. Immunoblot analyses, expression studies, measurement of Aβproteolytic fragments by ELISA, immunocytochemical and histologicalanalysis of brain sections suitable for use with the transgenic animalof the present invention are described herein. However, it will beunderstood by one of skill in the art that many other assays may also beused. The subject animals may be used by themselves, or in combinationwith control animals. Control animals may have, for example, wild-typeβAPP, tau, and/or presenilin transgenes that are not associated with AD,or may be transgenic for a control construct that does not contain aPresenilin, Tau or βAPP-encoding sequence. The screen using thetransgenic animals of the invention can employ any phenomena associatedwith AD or AD-related pathologies that can be readily assessed in ananimal model.

Therapeutic Agents

Once compounds have been identified in drug screening assays aseliminating or ameliorating the effects of AD and/or AD-relatedpathologies, these compounds can be used as therapeutic agents, providedthey are biocompatible with the animals, preferably humans, to whom theyare administered.

The therapeutic agents of the present invention can be formulated intopharmaceutical compositions by combination with appropriatepharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.Administration of the compounds can be administered in a variety of waysknown in the art, as, for example, by oral, buccal, rectal, parenteral,intraperitoneal, intradermal, transdermal, intratracheal, etc.,administration.

Depending upon the particular route of administration, a variety ofpharmaceutically acceptable carriers, well known in the art can be used.These carriers include, but are not limited to, sugars, starches,cellulose and its derivatives, malt, gelatine, talc, calcium sulfate,vegetable oils, synthetic oils, polyols, alginic acid, phosphatebuffered solutions, emulsifiers, isotonic saline, and pyrogen-freewater. Preservatives and other additives can also be present. Forexample, antimicrobial, antioxidant, chelating agents, and inert gasescan be added (see, generally, Remington's Pharmaceutical Sciences, 16thEdition, Mack, (1980)).

The concentration of therapeutically active compound in the formulationmay vary from about 0.1-100 wt. %.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific therapeutic agents, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given therapeutic agent are readily determinableby those of skill in the art by a variety of means. A preferred means isto measure the physiological potency of a given therapeutic agent.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used(e.g., amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Example 1 Materials and Methods

ELISAs and Immunoblots.

Aβ ELISAs were performed essentially as described previously (Suzuki, N.et al. (1994) An increased percentage of long amyloid beta proteinsecreted by familial amyloid beta protein precursor (beta APP717)mutants, Science 264, 1336-40). For immunoblots, brains extracted fromtransgenic and control mice were dounce homogenized in a solution of 2%SDS in H₂0 containing 0.7 mg/ml Pepstatin A supplemented with completeMini protease inhibitor tablet (Roche, No. 1836153). The homogenizedmixes were briefly sonicated to sheer the DNA and centrifuged at 4° C.for 1 hour at 100,000. The supernatant was used for immunoblot analysis.Proteins were resolved by SDS/PAGE (10% Bis-Tris from Invitrogen) underreducing conditions and transferred to nitrocellulose membrane. Themembrane was incubated in a 5% solution of not-fat milk for 1 hour at20° C. After overnight incubation at 4° C. with primary antibody, theblots were washed in tween-TBS for 20 minutes and incubated at 20° C.with secondary antibody. The blots were washed in T-TBS for 20 minutesand incubated for 5 minutes with Super Signal (Pierce).

Immunohistochemistry.

Formalin-fixed, paraffin-embedded brains were sectioned at 5 μm andmounted onto silane-coated slides and processed as described. Thefollowing antibodies were used: anti-Aβ 6E10 and 4G8 (SignetLaboratories, Dedham, Mass.), anti-Aβ 1560 (Chemicon), anti-APP 22C11(Chemicon), anti-Tau HT7, AT8, AT180 (Innogenetics), Tau C17 (SantaCruz), Tau 5 (Calbiochem), anti-GFAP (Dako), and anti-actin (Sigma).Primary antibodies were applied at dilutions of 1:3000 for GFAP, 1:1000for 6E10, 1:500 for 1560, AT8, AT180 and Tau5, and 1:200 for HT7.

Electrophysiology.

Mice were anaesthetized with halothane, decapitated and the brains wererapidly removed in ice-cold artificial cerebrospinal fluid (aCSF; 125 mMNaCl, 2.5 MM KCl, 1.25 mM KH2PO4, 25 mM NaHCO3, 1.2 mM MgSO4, 2 mMCaCl2, and 10 mM dextrose, bubbled with 95% O₂−5% CO₂, pH 7.4).Transverse hippocampal slices (400 μm) were prepared in aCSF using avibroslice, and left to equilibrate for at least 1 hour prior torecording in a holding chamber containing aCSF at room temperature.

Slices were placed in an interface chamber, continuously perfused withaCSF at 34° C. and covered with a continuous flow of warmed humidifiedgas (95% O₂, 5% CO₂). Field excitatory post-synaptic potentials (fEPSPs)were recorded in the stratum radiatum of the CA1 using glassmicroelectrodes (1-5Ω,˜5 μm diameter) filled with aCSF. Synapticresponses were evoked by stimulation of the Schaffercollateral/commissural pathway with a concentric bipolar stimulatingelectrode with 0.1 ms pulse-width. Input/output curves were generatedusing stimulus intensities from 0 to 300 μA in increments of 20 μA. PPFwas assessed using an interstimulus interval of 50 ms. Baseline fEPSPswere evoked at ˜30% of the max fEPSP for 15 minutes prior to highfrequency stimulation (HFS). LTP was induced at baseline intensity usingHFS consisting of 4 trains of 100 Hz stimulation at 20 s intervals.Recordings were made every 30 s for 60 minutes after HFS. The maximumfEPSP slopes were measured offline using Axograph software, and wereexpressed as a percentage of the average slope from the 15 minutes ofbaseline recordings. In some experiments the stimulus intensity wasraised so that baseline EPSP slopes matched the average baseline EPSP inthe NonTg mice (usually an EPSP ˜0.7 mV in amplitude). Data wereexpressed as mean±S.E.M, and assessed for significance using theStudent's t test.

Example 2 F₁ Hemizygous Triple Transgenic Mice

Generation of F₁ Transgenic Mice.

Presenilin-1 knockin (PS1_(M146V)KI) mice were provided by Dr. MarkMattson of the National Institutes of Health (NIH). The derivation andcharacterization of the PS1_(M146V)KI mice are described in Guo et al.(1999) Nat. Med. 5: 101-106; see also, Leissring, et al. (2000) J. CellBiol. 149: 793-797.

The triple transgenic mice were created by first harvesting single cellembryos from homozygous mutant presenilin-1 knockin mice (harboring theM146V mutation). Two transgenes were simultaneously microinjected intothese single cell embryos using the pronuclear microinjection technique.

The two transgenes contained cDNA molecules encoding either the humanSwedish APP mutation or the P301L mutation in the human tau gene. Bothof these cDNA molecules were separately inserted into the mouse Thy1.2gene cassette, allowing expression to be under the regulatory control ofthis sequence. The microinjected embryos were then transferred to fostermother mice and three weeks later pups were born.

These pups were screened to identify which ones contained the transgenesby Southern blot analysis. Five of the lines were found to contain bothtransgenes and were backcrossed to homozygous mutant presenilin-1knockin mice. By backcrossing the mice to homozygous mutant presenilin-1knockin mice, the mutant presenilin-1 transgene has been maintained on ahomozygous background and the mutant tau and APP transgenes on ahemizgygous background. The mice were subsequently bred such that allthree transgenes (presenilin-1, APP, and tau) are on a homozygosity, asdiscussed below.

The novel strategy used to generate the multi-transgenic mouse is shownin FIG. 1. Two mice transgenic for the mutant human gene presenilin-1knockin (PS1_(M146V))were mated and the resulting fertilized eggs(embryonic cells) harvested. A single embryonic cell wasco-microinjected with Thy1.2-APP_(SW695) and Thy1.2-TAU_(P301L), thenimplanted into a foster mother. The resulting offspring were founders ofa multi-transgenic (in this case, aPS1_(M146V)KI/APP_(SW695)/TAU_(P301L) triple-transgenic or “3×Tg-AD”)mouse line.

As will be appreciated, this process is a much simpler, faster andcost-effective method for producing a multi-transgenic mouse thanprevious breeding strategies, as the breeding and genotyping of themouse colony is greatly reduced.

Plasmids containing the tau(P301L) gene and the APPswe gene wereprovided by Dr. Michael Hutton and Dr. Rachel Neve, respectively. Thesequence for human β-amyloid precursor protein is found at GenBankAccession No. XM047793. The sequence for human tau gene is found atGenBank Accession No. 11426018.

The Thy1.2 gene was a gift from Dr. Pico Caroni. The sequence for theThy 1.2 gene is found at GenBank Accession No. M12379.

Founder Lines.

FIG. 2 shows a chart containing the relevant genotype for six founderlines resulting from the co-microinjection strategy described above andshown in FIG. 1. As indicated in FIG. 2, all of the founder lines arehomozygous for PS1_(M146V)KI, and five of the six lines, designated A2,B1, F5, F7, and G6, are hemizygous for both APP_(SWE) and Tau_(P301L).One of the founder lines, designated A1, is hemizygous only forTau_(P301L).

Expression Studies.

Immunoblots containing protein (80 μg) from three founder lines and onenon-transgenic control were probed with TauC17, an antibody that detectsboth mouse and human Tau protein, and HT7, an antibody specific forhuman Tau protein. TauC17 antibody was from Santa Cruz, Cat. No.SC-1995. The HT7 antibody was from Innogenetics, Cat. No. BR-01.

The results are shown in FIG. 3. As shown, two of the founder mouselines, A1 and B1, expressed human Tau protein (the double bands are dueto isoforms of the Tau protein). The G6 line also expresses low levelsof the human Tau protein, detected with this human specific protein, buthigh levels of the mouse tau protein (data not shown).

Similarly, as shown in FIG. 4, immunoblots containing protein from threefounder lines and one non-transgenic control were probed with 22C11, anantibody that detects both mouse and human APP protein, and 6E10, anantibody specific for human APP protein. The 22C11 antibody was fromChemicon International, Cat. No. MAB348. The 6E10 antibody is fromSignet, Cat. No. 9320-02. As is apparent, founder mouse line B1, shownto express human Tau protein in FIG. 3, also expressed human APPprotein. When full length APP protein is treated with β-secretase, aproteolytic fragment, C99, is produced that is the immediate precursorfor the highly amyloidogenic amyloid-62 (Aβ) peptide. In FIG. 5, proteinisolated from the brain of a B1 triple-transgenic 3×Tg-AD mouse and fromthe brain of a presenilin-1 knockin control mouse was probed with 6E10.The C99 fragment was found only in tissue from the B1 triple transgenicmouse, indicating that the B1 mouse both expressed and processed APPprotein from the human transgene.

This is further demonstrated in FIG. 6, wherein it is shown that a B1mouse (T13B1) also processed APP to Aβ. In FIG. 6A, a Western blotcontaining protein from T13B1 mice and from nontransgenic control micewere probed with a 1:500 dilution of primary antibody 6E10. The B1mouse, but not the control mouse, expressed the Aβ fragment of APP.

ELISA tests were used to measure the two isoforms of Aβ, namely, A▭ 40and Aβ42, in B1 mice and in transgenic control mice Tg2576, known toexpress high levels of Aβ. The Aβ 42 isoform is the more amyloidogenicof the two isoforms, and the ratio of Aβ 42 to Aβ 40 (42/40) isincreased in Alzheimer patients.

As shown in FIG. 6B, B1 mice express both Aβ isoforms, but have a higher42/40 ratio than the Tg2576 control mice, suggesting that the B1 tripletransgenic nice more closely exhibit traits consistent with Alzheimer'sdisease and, accordingly, provide an improved mouse model of thisdisease.

As discussed above, F₁ hemizygous triple transgenic mice were generatedby co-microinjecting two independent transgenes encoding human APP_(Swe)and human tau_(P301L), both under control of the mouse Thy1.2 regulatoryelement, into single-cell embryos harvested from homozygous mutantPS1_(M146V) knockin (KI) mice (FIG. 1). Genotype analysis by Southernblotting indicated that the tau and APP transgenes co-integrated at thesame locus, a finding further corroborated by analysis of thetransmission frequency in subsequent generations (data not shown).

Because the APP and tau transgenes are unlikely to independently assortand because the M146V mutation was “knocked in” to the endogenous mousePS1 locus 15, these 3×Tg-AD mice essentially breed as readily as a“single” transgenic line, even though the mice contain three transgenes.This facilitates the establishment and maintenance of the mouse colonyand reduces the need for genotypic analysis of the progeny. Moreover,this strategy results in another important pragmatic benefit as the3×Tg-AD mice are of the same genetic background, thereby eliminating aconfounding biological variable that is unavoidable when crossingindependent transgenic lines.

Example 3 Homozygous Triple Transgenic Mice

In order to generate mice homozygous for all three transgenes(PS1_(M146V), tau_(P301L), APP_(Swe)), hemizygous F1 mice were crossedto each other. FIG. 7 shows a southern blot identifying potentialcandidate homozygous mice. Homozygous mice have double the gene dosagecompared to hemizygous mice, as may be visualized by southern blotting.Such data may be confirmed by crossing the potential homozygous animalto a normal, nontransgenic mouse. If the mouse is homozygous, aspredicted by the southern blot, all of the offspring will be positivefor the transgenic trait. The results of such crosses are shown in thetable in FIG. 7. As indicated by the Comments section of the table, inwhich the number of positive transgenic mice identified and the numberof mice generated are shown, all of the candidate mice proved to behomozygous as 100% of their offspring were transgenic.

In FIG. 8A, protein from both hemizygous and homozygous B1 tripletransgenic mice, as well as from control presenilin-1 knockin singletransgenic mice, was probed with 6E10 antibody to human APP, HT7antibody to human Tau protein, or an antibody to β-actin (a control usedto ensure that equal amounts of protein from each mouse line were loadedonto the gel used to produce the immunoblot). As expected, both B1 mouselines expressed both Tau and APP proteins, with the homozygous B1 miceexpressing twice the amount of Tau and APP protein as the hemizygous B1mice. The presenilin-1 knockin mice show no Tau protein and only a smallamount of APP protein, resulting from cross-reactivity with mouse APPprotein.

A doubling of gene dosage is also apparent in selected mice by Southernblotting (FIG. 8B). Besides further facilitating the breeding andmaintenance of this mouse colony, the expression levels of human tau andAPP transgene products are doubled in the homozygous mice (FIG. 8C).Steady-state levels of both tau and APP approach about 3- to 4-fold andabout 6- to 8-fold endogenous levels in the brains of the hemizygous andhomozygous mice, respectively (FIG. 8D). The maintenance of bothhemizygous and homozygous 3×Tg-AD mice renders it possible to study theeffect these gene interactions exert as a function of age in a contextin which the expression levels are doubled in mice of the same geneticbackground.

Example 4 Pathological Alterations in the Triple Transgenic Mice

Sections from the brains of B1 triple transgenic mice and PS1-KI control(single transgenic) nice were prepared and stained with hematoxylin andeosin. The brain section from B1 mice (FIG. 9) exhibits a tangle-likepathology, indicated by the arrows, similar to that seen in the brainsof Alzheimer patients. This pathological alteration is not seen in thebrain sections taken from PS1-KI control mice.

Brain sections from the triple transgenic B1 mice also exhibit Aβdeposition, a common feature in the brains of Alzheimer patients. InFIG. 10, brain sections from B1 transgenic mice stained with 6E10antibody to human APP show Aβ deposition.

Example 5 Analysis of Steady-State Levels of Human Transgenes in 3xTL-ADMice

The mouse Thy1.2 expression cassette has been demonstrated to drivetransgene expression predominantly to the CNS (Caroni, P. (1997)Overexpression of growth-associated proteins in the neurons of adulttransgenic mice, J. Neurosci. Methods 71, 3-9). Immunoblot analysis forhuman APP and tau in multiple peripheral tissues from the B13×Tg-AD line(the highest expressing line characterized) confirmed that expressionwas predominantly, if not exclusively, restricted to the CNS (FIG. 11A).To determine which regions of the CNS expressed the human APP and tauproteins, multiple brain regions (hippocampus, cortex, cerebellum, etc.)were microdissected, protein extracts were prepared and steady-statelevels of the transgenic human proteins were measured by westernblotting (FIG. 11B). As shown, the AD-relevant regions, including thehippocampus and cerebral cortex, were among the regions containing thehighest steady-state levels of both the transgene-derived human APP andtau proteins. Other regions, such as the cerebellum, did not appear tocontain any transgenic proteins, either because they are not expressedthere or are rapidly degraded.

To determine whether the APP protein was processed to liberate the Aβfragment, brain homogenates from hemizygous and homozygous mice wereanalyzed by immunopreciptation/western blotting. FIG. 11C shows thepresence of a 4-kDa species after probing with an Aβ-derived antibodythat is twice as abundant in the homozygous versus the hemizygousbrains, but undectectable in NonTg brain homogenates. Aβ40 and Aβ42levels were also compared in the brains of age- and sex-matched NonTgand hemizygous and homozygous 3×Tg-AD mice by ELISA. There is aprogressive increase in Aβ formation as a function of age in the 3×Tg-ADbrains, and a particularly dramatic effect on Aβ42 levels (FIG. 11D).

Example 6 Aβ Deposition Precedes Tau Pathology in 3×Tg-AD Mice

Intraneuronal Aβ immunoreactivity is one of the earliestneuropathological manifestations in the 3×Tg-AD mice, first detectablein neocortical regions, and subsequently in CA1 pyramidal neurons.Intracellular Aβ immunoreactivity is apparent between 3-4 months of agein the neocortex of 3×Tg-AD mice, and by 6 months of age in the CA1subfield of the hippocampus of hemizygous and homozygous mice (FIG.12A,D). This is followed by the emergence of extracellular Aβ depositsseveral months later (FIG. 12B,C). As with the intraneuronal staining,the largest cluster of extracellular Aβ deposits are first found in thefrontal cortex and occur predominantly in layers 4-5, but involves othercortical layers and the hippocampus in older mice. By 12 months,extracellular Aβ deposits are readily apparent in other corticalregions, suggesting that there is an age-related, regional dependence tothe Aβ deposits in the 3×Tg-AD mice that closely mimics the patternfound in the human AD brain. The progressive increase in Aβ depositionis corroborated by the ELISA data (FIG. 11D).

Because of the approach used to generate the 3×Tg-AD mice, both the tauand APP transgenes are expressed to comparable levels in the same brainregions. Consequently, these mice can be used to directly test theamyloid cascade hypothesis, which predicts that Aβ is the initiatingtrigger that underlies all cases of AD (Hardy, J. & Selkoe, D. J. (2002)The amyloid hypothesis of Alzheimer's disease: progress and problems onthe road to therapeutics, Science 297,353-56). The development of Aβ andtau pathology in 3×Tg-AD mice, ranging in age from 6 to 15 months, wascompared (cf FIG. 12 d-f and 12 g-i). Whereas intracellular Aβimmunoreactivity is present by 6 months of age in the hippocampus (andextracellular Aβ deposits in cortex), no significant tauimmunoreactivity is present at this age (FIG. 12 g). Human tauimmunoreactivity is first evident at 12 months of age in the CA1neurons, showing extensive immunolabeling of the somatodendriticcompartments, and progressively more staining by 15 months (FIG. 12H,I).

Example 7 Tau Pathology Initiates in the Hippocampus and Progresses tothe Neocortex

A subset of the neurons that are immunopositive for the human-specifictau antibody HT7 are also immunoreactive with several conformational-and phosphotau-specific antibodies. These include the conformationspecific antibody MC1 (FIG. 13A,B), AT8, which detects tauphosphorylated at serine 202 and threonine 205 (FIG. 13C), and AT180,which detects phosphorylated tau at threonine 231 (FIG. 13D,E). Asshown, tau is aberrantly hyperphosphorylated at multiple residues in thebrains of the 3×Tg-AD mice. In contrast to the Aβ staining, tauimmunostaining was first apparent in the CA1 region of the hippocampusand then progressively involved cortical neurons (FIG. 13F).Histological stains such as Gallayas and thioflavin S can also identifytau-reactive neurons (FIG. 13G,H). Finally, it is noted that reactiveastrocytes were also readily apparent adjacent to extracellular Aβdeposits (FIG. 13J).

The 3×Tg-AD mice develop a progressive and age-dependent Aβ and taupathology. The analysis presented herein indicates that Aβ pathologyprecedes tau pathology. As has been reported previously, it is likelythat Aβ pathology affects the development of tau pathology (Lewis, J. etal. (2001) Enhanced neurofibrillary degeneration in transgenic miceexpressing mutant tau and APP, Science 293, 1487-91; Gotz, J., et al.(2001) Formation of neurofibrillary tangles in P3011 tau transgenic miceinduced by Aβ 42 fibrils, Science 293, 1491-5). Although Aβ and taupathology initiate in different brain regions in the 3×Tg-AD mice (i.e.,cortex for Aβ and hippocampus for tau), it is not inconsistent with thenotion that Aβ influences tau pathology. The hypothesis that Aβinfluences tau pathology is further supported by the finding that tauand Aβ immunoreactivity may be co-localized to the same neurons asrevealed by double-labeling immunohistochemistry (FIG. 13I).Consequently, it is likely that intracellular Aβ immunoreactivity (whichis the first detected pathological manifestation) affects thedevelopment of the tau pathology.

Example 8 Age Related Synaptic Dysfunction in 3×Tg-AD Mice

Neuronal and synaptic dysfunction are major phenotypic manifestations ofAD neuropathology. Synaptic dysfunction, for example, is among the bestcorrelates for the memory and cognitive changes that characterize AD.One- and six-month old homozygous 3×Tg-AD mice were compared todetermine if there was an age-related impairment in synaptic functioningin the CA1 hippocampal region. Age- and sex-matched NonTg andPS1_(M146V) KI mice were used as controls. PS1_(M146V) KI mice wereevaluated and used as controls because the electrophysiologicalproperties of this line were unknown and because the 3×Tg-AD mice weredirectly derived from this line. Thus it was crucial to determine theeffect of the PS1 mutation on synaptic functioning. In addition, the6-month time point was selected because extracellular Aβ deposits areevident only in the neocortex and not in the CA1 region of thehippocampus, although intracellular Aβ immunoreactivity is apparent.This allowed us to determine whether synaptic dysfunction precedesplaque and tau pathology in these nice.

To investigate basal synaptic transmission, input/output (I/O) curveswere generated by measuring field excitatory postsynaptic potentials(fEPSPs) elicited in CA1 by stimulation of the Schaffer collaterals atincreasing stimulus intensities. The I/O curves between one monthPS1_(M146V) KI and 3×Tg-AD mice were not significantly different fromNonTg mice (FIG. 14A). In contrast, 6 month PS1_(M146V) KI and 3×Tg-ADmice exhibited lesser fEPSP slopes and amplitudes at all stimulusintensities tested, and had significantly reduced maximum fEPSPsrelative to NonTg mice (FIG. 14 B). The PS1_(M146V) KI mice, however,were not significantly different from the 3×Tg-AD mice (P<0.1). Theseresults show that basal synaptic transmission is impaired in both thePS1_(M146V) KI and 3×Tg-AD mice at 6 months of age.

Paired-pulse facilitation (PPF), a measure of short-term plasticity, wasalso measured. No differences were observed in 1-month old PS1_(M146V)KI and 3×Tg-AD mice (FIG. 14 C). At 6-months of age there was nodifference in the amount of facilitation between NonTg and 3×Tg-AD mice(NonTg, 31 ±1.5%; 3×Tg-AD, 30 ±2.7%, P<0.5), but PS1_(M146V) KI miceexhibited significantly enhanced PPF compared to NonTg mice (43 ±2.9%,P<0.01; FIG. 14D). The mechanisms underlying PPF are thought to bepresynaptic (Zucker, R. S. & Regehr, W. G. (2002) Short-term synapticplasticity, Annu. Rev. Physiol. 64,355-405) and probably involveresidual Ca²⁺ in the nerve terminal after the first stimulus, leading toincreased neurotransmitter release during the second stimulus (Thomson,A. M. (2002) Facilitation, augmentation and potentiation at centralsynapses, Trends Neurosci. 23, 305-12). The results shown herein suggestthat presynaptic mechanisms are intact in 3×Tg-AD mice, and theenhancement of facilitation in the PS1_(M146V) KI mice may be due toalterations in handling intracellular Ca²⁺ (Leissring, M. A. et al.(2000) Capacitative calcium entry deficits and elevated luminal calciumcontent in mutant presenilin-1 knockin mice, J. Cell Biol. 149, 793-8;LaFerla, F. M. (2002) Calcium dyshomeostasis and intracellular signalingin Alzheimer's disease (2002) Nat. Reviews Neurosci. 3, 862-872).

Long-term potentiation (LTP), a form of plasticity thought to underlielearning and memory (Bliss, T. V. & Collingridge, G. L. (1993) Asynaptic model of memory: long-term potentiation in the hippocampus,Nature 361, 31-9), was investigated in the CA1 hippocampal region. Nodifferences were observed between lines at 1-month of age (FIG. 14E).LTP in 6-month old 3×Tg-AD mice was also investigated, and initialexperiments showed that LTP was severely impaired in the 3×Tg-AD mice.However, because the LTP induction protocol used a stimulus intensitythat elicited ˜30% of the maximum fEPSP slope during baseline and highfrequency stimulation (HFS), it is plausible that the smaller absolutefEPSP may account for the LTP deficits.

To address this possibility, the stimulus intensity was adjusted tomatch baseline fEPSP magnitudes to those of NonTg mice. LTP magnitudesin these experiments did not significantly differ when weaker stimulusintensities were used (data not shown), indicating that reduced basaltransmission does not likely account for the deficits in LTP, although aceiling affect cannot be discounted. All the data from the 3×Tg-AD micewas subsequently pooled and demonstrated reduced LTP (FIG. 14F). Incontrast, LTP in the PS1_(M146V) KI mice was essentially normal and didnot differ from NonTg mice 50-60 minutes after HFS, despite weakerfEPSPs relative to NonTg controls. There was, however, a trend in thePS1_(M146V) KI mice for significantly higher LTP during the first 10minutes after HFS, as has also been reported in the transgenic miceoverexpressing PS1_(A246E) variant (Parent, A., supra). As with the3×Tg-AD mice, raising baseline fEPSPs to NonTg levels did not result insignificantly different LTP magnitude and thus the data were pooled.

These results show that the 3×Tg-AD mice exhibit deficits in basalsynaptic transmission and LTP with the appearance of intracellular Allbut prior to the formation of extracellular plaques and tau pathology.In contrast, the PS1_(M146V) KI mice demonstrated normal-to-enhancedLTP, despite deficits in basal synaptic transmission, suggesting thatthe mechanisms underlying the transmission and LTP deficits in the3×Tg-AD mice may be independent. All the groups compared in thesestudies have the same genetic background, which helps analysisconsiderably as LTP can be markedly affected in different inbred mousestrains (Nguyen, P. V., et al. (2000) Strain-dependent differences inLTP and hippocampus-dependent memory in inbred mice, Learn. Mem. 7,170-9). The subsequent characterization of synaptic dysfunction in thesetwo models (i.e., 3×Tg-AD and PS1_(M146V) KI) will help resolve theunderlying mechanisms involved and the contribution of the differentgene mutations that have been implicated in AD. In addition, futurestudies will help discern the relationship that Aβ and tau pathologyexert on synaptic function.

A novel strategy to develop multi-transgenic animals, and in particulara triple transgenic model of AD, has been disclosed herein. Compared tocross-breeding, this approach has several major advantages. The APP andtau transgenes co-integrated at the same genetic locus, rendering itunlikely that either transgene will independently assort in subsequentgenerations. Consequently, this tight linkage coupled to the ‘knockin’of the PS1 mutation indicates that the 3×Tg-AD mice breed as readily asany single transgenic line, particularly since these mice have also beenbred to homozygosity. Thus, deriving large numbers of these 3×Tg-AD miceis straightforward and cost-effective. Moreover, the easy maintenance ofthis transgenic line facilitates the crossing of these 3×Tg-AD mice toother transgenic or gene-targeted mice. Finally, another major advantagewith this approach is that multiple transgenes are introduced into ananimal without altering or mixing the background genetic constitution.Thus, an important confounding variable is avoided, which may be acrucial issue for behavioral, electrophysiological, and vaccine-basedexperiments.

As shown, 3×Tg-AD mice develop an age-related and progressiveneuropathological phenotype that is more robust in the homozygous mice.Intracellular Aβ immunoreactivity is the first clear neuropathologicalmanifestation in the brains of these transgenic mice. There is evidencethat intracellular Aβ deposition may be important in the pathogenesis ofAD (LaFerla, F. M., et al. (1997) Neuronal cell death in Alzheimer'sdisease correlates with apoE uptake and intracellular Aβ stabilization,J. Clin. Invest. 100, 310-20; Gouras, G. K et al. (2000) IntraneuronalAβ42 accumulation in human brain, Am. J. Pathol. 156, 15-20) and in therelated disorder inclusion body myositis (Sugarman, M. C. et al. (2002)Inclusion body myositis-like phenotype induced by transgenicoverexpression of βAPP in skeletal muscle, Proc. Natl. Acad. Sci. U.S.A.99, 6334-9; Mendell, J. R., et al. (1991) Amyloid filaments in inclusionbody myositis: Novel findings provide insight into nature of filaments,Arch. Neurol. 48, 1229-34). Intraneuronal Aβ has also been documented inthe brains of other AD transgenic mouse models (Wirths, O. et al. (2001)Intraneuronal Aβ accumulation precedes plaque formation in beta-amyloidprecursor protein and presenilin-1 double-transgenic mice, Neurosci.Lett. 306, 116-20; Li, Q. X. et al. (1999) Intracellular accumulation ofdetergent-soluble amyloidogenic Aβ fragment of Alzheimer's diseaseprecursor protein in the hippocampus of aged transgenic mice, J.Neurocheni. 72, 2479-87; Kuo, Y. M. et al. (2001) The evolution of Aβpeptide burden in the APP23 transgenic mice: implications for Aβdeposition in Alzheimer disease, Mol. Med. 7, 609-18; LaFerla, F. M., etal. (1995) The Alzheimer's Aβ peptide induces neurodegeneration andapoptotic cell death in transgenic mice, Nat. Genet. 9, 21-30). Thepathophysiological relevance of intraneuronal Aβ, however, is not yetresolved, although it is tempting to speculate that it maybe the sourceof the extracellular Aβ deposits. Herein it is shown that the occurrenceof intraneuronal Aβ in CA1 pyramidal neurons correlates with impairmentsin synaptic transmission, including deficits in LTP. The finding thatsynaptic transmission and LTP deficits precede overt plaque and taupathology suggests that synaptic dysfunction is an early manifestationof AD. Furthermore, these studies indicate that extracellular Aβdeposition is not the causal factor underlying the synaptic dysfunction.This agrees well with recent findings that show that soluble Aβoligomers inhibit LTP in vivo (Walsh, D. M. et al. (2002) Naturallysecreted oligomers of amyloid beta protein potently inhibit hippocampallong-term potentiation in vivo, Nature 416, 535-9).

It is also shown herein that Aβ pathology precedes tau pathology in thismodel. Because the APP and tau transgenes were expressed to comparablelevels, this observation provides strong experimental support for theamyloid cascade hypothesis. This hypothesis posits that Aβ accumulation,which may occur as a consequence of overproduction, mismetabolism, orfailures in clearance, is the initiating trigger that underlies allforms of AD. The development of both Aβ and tau pathology in the 3×Tg-ADmouse model is significant as it should enable a more accurateevaluation of potential therapeutic interventions (such as Aβimmunizations) in an animal model that more closely mimics theneuropathology of AD. In addition, this model will be useful fordetermining if modulation of either the Aβ or tau pathology impacts thedevelopment of the other.

While this invention has been described in detail with reference to acertain preferred embodiments, it should be appreciated that the presentinvention is not limited to those precise embodiments. Rather, in viewof the present disclosure which describes the current best mode forpracticing the invention, many modifications and variations wouldpresent themselves to those of skill in the art without departing fromthe scope and spirit of this invention. In particular, it is to beunderstood that this invention is not limited to the particularmethodology, protocols, cell lines, animal species or genera,constructs, and reagents described as such may vary, as will beappreciated by one of skill in the art. The scope of the invention is,therefore, indicated by the following claims rather than by theforegoing description. All changes, modifications, and variations comingwithin the meaning and range of equivalency of the claims are to beconsidered within their scope.

1. A triple transgenic animal whose genome comprises: a first transgene,encoding human presenilin-1 polypeptide having a mutation associatedwith Alzheimer's disease (AD) or AD-type pathology, wherein the firsttransgene is operably linked to a first promoter, a second transgene,encoding human Tau protein having a mutation associated with AD orAD-type pathology, wherein the second transgene is operably linked to asecond promoter, and a third transgene, encoding human β-amyloidprecursor protein (βAPP) having a mutation associated with AD or AD-typepathology, wherein the transgene is operably linked to a third promoter,wherein expression of the transgenes results in a Tau pathology in thetriple transgenic animal.
 2. The animal of claim 1, wherein the firsttransgene encodes PS1_(M146V).
 3. The animal of claim 1, wherein thesecond transgene encodes Tau_(P 301L).
 4. The animal of claim 1, whereinthe third transgene encodes APP_(SWE).
 5. The animal of claim 1, whereinthe first promoter is an endogenous presenilin promoter.
 6. The animalof claim 1, wherein the second promoter is a Thy1.2 promoter.
 7. Theanimal of claim 1, wherein the third promoter is a Thy1.2 promoter. 8.The transgenic animal of claim 1, wherein the animal is fertile andtransmits the three transgenes to its offspring.
 9. The transgenicanimal of claim 1, wherein the second and third transgenes have beenintroduced into an ancestor of said animal at an embryonic stage. 10.The transgenic animal of claim 1, wherein the animal is hemizygous forthe human βAPP transgene.
 11. The transgenic animal of claim 1, whereinthe animal is homozygous for the human βAPP transgene.
 12. Thetransgenic animal of claim 1, wherein the animal is hemizygous for thehuman tau transgene.
 13. The transgenic animal of claim 1, wherein theanimal is homozygous for the human tau transgene.
 14. The transgenicanimal of claim 1, wherein the animal is a mouse.
 15. A tripletransgenic animal whose genome comprises: a first transgene, encodinghuman presenilin-1 polypeptide having a mutation associated with AD orAD-type pathology, wherein the first transgene is operably linked to afirst promoter, a second transgene, encoding human tau protein having amutation associated with AD or AD-type pathology, wherein the secondtransgene is operably linked to a second promoter, and a thirdtransgene, encoding human β-amyloid precursor protein having a mutationassociated with AD or AD-type pathology, wherein the transgene isoperably linked to a third promoter, wherein expression of thetransgenes results in an Aβ pathology in the triple transgenic animal.16. The animal of claim 15, wherein the first transgene encodesPS1_(M146V) .
 17. The animal of claim 15, wherein the second transgeneencodes Tau_(P301L).
 18. The animal of claim 15, wherein the thirdtransgene encodes APP_(SWE).
 19. The animal of claim 15, wherein thefirst promoter is an endogenous presenilin promoter.
 20. The animal ofclaim 15, wherein the second promoter is a Thy1.2 promoter.
 21. Theanimal of claim 15, wherein the third promoter is a Thy1.2 promoter. 22.The transgenic animal of claim 15, wherein the animal is fertile andtransmits the three transgenes to its offspring.
 23. The transgenicanimal of claim 15, wherein the second and third transgenes have beenintroduced into an ancestor of said animal at an embryonic stage. 24.The transgenic animal of claim 15, wherein the animal is hemizygous forthe human βAPP transgene.
 25. The transgenic animal of claim 15, whereinthe animal is homozygous for the human βAPP transgene.
 26. Thetransgenic animal of claim 15, wherein the animal is hemizygous for thehuman tau transgene.
 27. The transgenic animal of claim 15, wherein theanimal is homozygous for the human tau transgene.
 28. The transgenicanimal of claim 15, wherein the animal is a mouse.
 29. A method formaking a multi-transgenic animal, the method comprising: providing atransgenic animal homozygous for a first transgene; microinjecting asecond transgene into an embryo of the transgenic animal; and allowingthe embryo to develop to term to produce the multi-transgenic animal.30. The method of claim 29, further comprising the step ofmicroinjecting at least a third transgene into the embryo.
 31. A methodfor making a triple transgenic animal, the method comprising: providinga transgenic animal homozygous for a first transgene; co-microinjectinga second transgene and a third transgene into the transgenic animal; andallowing the embryo to develop to term to produce the triple transgenicanimal.
 32. A multi-transgenic animal, produced by the steps of:providing a transgenic animal homozygous for a first transgene;microinjecting a second transgene into an embryo of the transgenicanimal; and allowing the embryo to develop to term to produce themulti-transgenic animal.
 33. A triple transgenic animal, produced by thesteps of: providing a transgenic animal homozygous for a firsttransgene; co-microinjecting a second transgene and a third transgeneinto the transgenic animal; and allowing the embryo to develop to termto produce the triple transgenic animal.
 34. A triple transgenic animalwhose genome comprises: a first transgene, encoding human presenilin-1polypeptide having a mutation associated with AD or AD-type pathology,wherein the first transgene is operably linked to a first promoter, asecond transgene, encoding human Tau protein having a mutationassociated with AD or AD-type pathology, wherein the second transgene isoperably linked to a second promoter, and a third transgene, encodinghuman β-amyloid precursor protein (βAPP) having a mutation associatedwith AD or AD-type pathology, wherein the transgene is operably linkedto a third promoter, wherein expression of the transgenes results in aTau pathology and an AP pathology in the triple transgenic animal.
 35. Atriple transgenic animal whose genome comprises: a first transgene,encoding human presenilin-1 polypeptide having a mutation associatedwith AD or AD-type pathology, wherein the first transgene is operablylinked to a first promoter, a second transgene, encoding human Tauprotein having a mutation associated with AD or AD-type pathology,wherein the second transgene is operably linked to a second promoter,and a third transgene, encoding human β-amyloid precursor protein (βAPP)having a mutation associated with AD or AD-type pathology, wherein thetransgene is operably linked to a third promoter, wherein expression ofthe transgenes results in an AD or AD-type pathology in the tripletransgenic animal.
 36. A method of screening biologically active agentsthat facilitate reduction of a tau pathology in vivo, the methodcomprising: administering a candidate agent to a transgenic animalaccording to claim 1, and determining the effect of said agent on theTau pathology.
 37. A method of screening biologically active agents thatfacilitate reduction of an Aβ pathology in vivo, the method comprising:administering a candidate agent to a transgenic animal according toclaim 15, and determining the effect of said agent on the Aβ pathology.38. A method of screening biologically active agents that facilitatereduction in both Tau pathology and Aβ pathology in vivo, the methodcomprising: administering a candidate agent to a transgenic animalaccording to claim 34, and determining the effect of said agent on bothTau pathology and AP pathology.
 39. A method of screening biologicallyactive agents that facilitate reduction in AD or AD-type pathology invivo, the method comprising: administering a candidate agent to atransgenic animal according to claim 35, and determining the effect ofsaid agent on the AD or AD-type pathology.
 40. A composition fortreating AD or AD-type pathology in a patient, wherein the compositionhas been screened by the method of claim 39 and found to be effective inreducing AD or AD-type pathology.
 41. A method of treating a patientdiagnosed with AD, comprising administering to the patient atherapeutically effective amount of the composition of claim 40.